27.1: Classification of Amino Acids.Chapter 27: Amino Acids, Peptides, and Proteins. monomer unit:...
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Chapter 27: Amino Acids, Peptides, and Proteins.monomer unit: α-amino acids
Biopolymer: the monomeric amino acids are linked through an amide bond (the carboxylic acids of one AA with the α-amino group of a second)
peptide (< 50 amino acids)protein (> 50 amino acids)
R CO2H
NH2H
!- Amino Acid
R = sidechain
R1
H3N CO2
R2
H3N CO2
+ - H2OR1
H3N
HN
O
CO2
R2
C-terminus
N-terminus
Peptide or protein (polypeptide)
NH
O
R3
HN
O
R2
NH
O
R1
HN
NH
O
R7
HN
O
R6
NH
O
R5
R4 HN
O
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27.1: Classification of Amino Acids. AA’s are classified according to the location of the amino group.
There are 20 genetically encoded α-amino acids found in peptides and proteins
19 are primary amines, 1 (proline) is a secondary amine19 are “chiral”, 1 (glycine) is achiral; the natural configuration of the α-carbon is L.
!-amino acid(2-amino carboxylic acid)
C CO2HH2N
H
H
C CO2HC
H
H
H2N
H
H
C CC
H
H
H2N
H
H
CO2H
H
H
"-amino acid(3-amino carboxylic acid)
#-amino acid(4-amino carboxylic acid)
CO2H
CH3
H2N H
CO2H
R
H2N H
L-alanine
CHO
HO H
H OH
CH2OH
CHO
H OH
HO H
CH2OH
L-erythroseD-erythrose
CO2H
H2N H
H OH
CH3
CO2H
H2N H
H3C H
CH2CH3
L-theronine(2S,3R)
L-isoleucine(2S,3S)
CHO
CH2OH
H OH
D-glyceraldehyde
CHO
CH2OH
HO H
L-glyceraldehyde

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α-Amino acids are classified by the properties of their sidechains. Nonpolar:
(S)-(+)-Valine (Val, V)
(S)-(–)-Tryptophan (Trp, W)(S)-(–)-Proline (Pro, P) (S)-(–)-Phenylalanine (Phe, F)
COO–
NH3
H
N
H
COO–
NH3
COO–
NH3
COO–
NH3
NH3
COO–
COO–
(2S,3S)-(+)-Isoleucine (Ile, I)
(S)-(+)-Alanine (Ala, A)
(S)-(–)-Leucine (Leu, L)
Glycine (Gly, G)
NH3
COO–
COO–
NH3NH
NH3
COO–S
(S)-(–)-Methionine (Met, M)
Polar but non-ionizable:
(S)-(+)-Glutamine (Gln, Q)
H2N
O
NH3
COO–
(S)-(–)-Tyrosine (Tyr, Y)(2S,3R)-(–)-Threonine (Thr, T)(S)-(–)-Serine (Ser, S)
COO–
NH3
COO–
NH3
COO–
NH3 NH3
COO–
NH3
COO–
O
H2NHS
HO
OH
HO
(R)-(–)-Cysteine (Cys, C) (S)-(–)-Asparagine (Asn, N)
pKa ~ 13 pKa ~ 13
pKa ~ 8.2
pKa ~ 10.1
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Acidic:
Basic:
(S)-(–)-Histidine (His, H)
NH3
COO–COO–
NH3 NH3
COO–N
H
NN
H
H
(S)-(+)-Lysine (Lys, K) (S)-(+)-Arginine (Arg, R)
pKa ~ 10.5 pKa ~ 6.0 pKa ~ 12.5
H2N
NH H
H3N
-O
O NH3
COO–
(S)-(+)-Aspartic Acid (Asp, D)
COO–
NH3
O
-O
(S)-(+)-Glutamic Acid (Glu, E)
pKa ~ 3.6 pKa ~ 4.2
27.2: Stereochemistry of Amino Acids: The natural configuration of the α-carbon is L. D-Amino acids are found in the cell walls of bacteria. The D-amino acids are not geneticallyencoded, but derived from the epimerization of L-isomers

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27.3: Acid-Base Behavior of Amino Acids. Amino acids exist as a zwitterion: a dipolar ion having both a formal positive and formal negative charge (overall charge neutral).
CO2H
R
H
H2N CO2
R
H
H3N_+
Amino acids are amphoteric: they can react as either an acid or abase. Ammonium ion acts as an acid, the carboxylate as a base.
Isoelectric point (pI): The pH at which the amino acid exists largely in a neutral, zwitterionic form (influenced by the nature of the sidechain)
pKa ~ 5 pKa ~ 9
CO2
R
H
H3N_+CO2H
R
H
H3N+
CO2
R
H
H2NHO
_
pKa2low pH high pH
_H3O+
pKa1
Table 27.2 (p. 1115) & 27.2 (p. 1116)
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pI =pKax + pKay
2
CO2
CH3
H
H3NCO2H
CH3
H
H3N
low pH
CO2
CH3
H
H2N+ +
high pH
pKa1(2.3)
pKa2(9.7)
CO2H
CH2
H
H3N
CO2H
pKa3(3.6)
CO2
H
H3N
CH2
CO2H
CO2
H
H3N
CH2
CO2
pKa2(9.6)
pKa1(1.9)
CO2
H
H2N
CH2
CO2
low pH high pH
CO2H
(CH2)4
H
H3N
NH3
pKa2(9.0)
CO2
H
H3N
(CH2)4
NH3
CO2
H
H2N
(CH2)4
NH3
pKa3(10.5)
pKa1(2.2)
CO2
H
H2N
(CH2)4
NH2
low pH high pH

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Electrophoresis: separation of polar compounds based on theirmobility through a solid support. The separation is based on charge (pI) or molecular mass.
+ _
_ _ _ _ + + + +
+ _
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Strecker Synthesis: recall reductive amination
C CO2HR
O NH3
C CO2HR
NH2
H
NaB(CN)H3
R C CO2H
NH2
H
C HR
O NH3
C HR
NH2NaC!N
N!C:
R C C!N
NH2
H
H3O+
-or-NaOH, H2O
R C CO2H
NH2
H
Amidomalonate Synthesis: recall the malonic acid synthesis
H CO2EtC
CO2EtHN EtO Na
RCH2X
H3O
- CO2 RCH2 CO2HC
HH2N
O
RCH2 CO2EtC
CO2EtHN
O
R-CH2-CO2H R C CO2H
Br
R C CO2H
NH2Br2, PBr3 NH3
H H
27.5: Synthesis of Amino Acids:
Ch. 19.16

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R CO2
H3N H H3C O CH3
O O
base R CO2H
HN H
O
H3C
HOCH2CH3
H+R CO2CH2CH3
H2N H
27.6: Some Biochemical Reactions of Amino Acids. Manyenzymes involved in amino acid biosynthesis, metabolism and catabolism are pyridoxal phosphate (vitamin B6) dependent (please read)
NH3
R CO2-
H
L-amino acid
NH3
R HCO2
-
D-amino acid
racemase,epimerase
H3N
RH
H
decarboxylase
O
R CO2-
transaminase
N
2-O3POOH
O H
pyridoxalphosphate (PLP)
+
NH
PO
N
OH
CO2-R
27.5: Reactions of Amino Acids. Amino acids will undergoreactions characteristic of the amino (amide formation) and carboxylic acid (ester formation) groups.
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27.7: Peptides. Proteins and peptides are polymers made up of amino acid units (residues) that are linked together through theformation of amide bonds (peptide bonds) from the aminogroup of one residue and the carboxylate of a second residue
By convention, peptide sequences are written left to right from the N-terminus to the C-terminus
H2N CO2H
Alanine
H2N CO2H
HO
Serine
+ - H2O
H2N
HN
O
CO2H
OH
Ala - Ser(A - S)- H2O
H2N
HN
O
CO2H
HO
Ser - Ala(S - A)
C-terminus
N-terminus
C-terminus
N-terminus
NH
O
R3
HN
O
R2
NH
O
R1
HN
NH
O
R7
HN
O
R6
NH
O
R5
R4 HN
O
backbone

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The amide (peptide) bond has C=N double bond character dueto resonance resulting in a planar geometry
restricts rotationsresistant to hydrolysis
R1
HN
N
O R2 HN
O
amide bond
R1
HN
N
O R2 HN
O
_
+
H H
The N-H bond of one amide linkage can form a hydrogen bondwith the C=O of another.
N
N
O
H
R
O
H
N
N
O
H
R
O
H
N
N
O
H
R
O
H
N-O distance 2.85 - 3.20 Å optimal N-H-O angle is 180 °
Disulfide bonds: the thiol groups of cysteine can be oxidized to form disulfides (Cys-S-S-Cys)
SHHO2C
NH22
1/2 O2H2O
SHO2C
NH2
SCO2H
NH2H2
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NH
O
R2
HN
O
R1
NH
HN
O
R5
NH
O
R4
HN
O
NH
OHN
O
R6
NH
HN
O
R10
NH
O
R9
R8 HN
O
SH
NH
O
R2
HN
O
R1
NH
HN
O
R5
NH
O
R4
HN
O
NH
OHN
O
R9
NH
HN
O
R13
NH
O
R12
R11 HN
O
S
SHS 1/2 O2
H2
1986 Nobel Prize in Medicine:Stanley Cohen Rita Levi-Montalcini
Epidermal Growth Factor (EGF): the miracle of mother’s spit 53 amino acid, 3 disulfide linkages

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27.8: Introduction to Peptide Structure Determination.Protein Structure:primary (1°) structure: the amino acid sequencesecondary (2°): frequently occurring substructures or foldstertiary (3°): three-dimensional arrangement of all atoms in a
single polypeptide chainquaternary (4°): overall organization of non-covalently linked
subunits of a functional protein.
1. Determine the amino acids present and their relative ratios2. Cleave the peptide or protein into smaller peptide fragments
and determine their sequences3. Cleave the peptide or protein by another method and
determine their sequences. Align the sequences of thepeptide fragments from the two methods
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E-A-Y-L-V-C-G-E-RF-V-N-Q-H-L-F-S-H-L-KG-C-F-L-P-KL-G-A
F-V-N-Q-H-L-FS-H-L-K-E-A-YL-V-C-G-E-R-G-C-FL-P-K-L-G-A
F-V-N-Q-H-L-F F-V-N-Q-H-L-F-S-H-L-K S-H-L-K-E-A-Y E-A-Y-L-V-C-G-E-R L-V-C-G-E-R-G-C-F G-C-F-L-P-K L-P-K-L-G-A
L-G-A
F-V-N-Q-H-L-F-S-H-L-K-E-A-Y-L-V-C-G-E-R-G-C-F-L-P-K-L-G-A

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27.9: Amino Acid Analysis. automated method to determine the amino acid content of a peptide or protein
Reaction of primary amines with ninhydrin
peptide-or-
protein
[H] reduce anydisulfidebonds
Enzymaticdigestion
R CO2
NH3 individualamino acids-or-
H3O+, Δ
1972 Nobel Prize in ChemistryWilliam Stein Stanford Moore
liquidchromatography
derivatize w/ninhydrin
Detected w/UV-vis
Different amino acids have different chromatographicmobilities (retention times)
R CO2
NH3
O
O
O+
Ninhydrin
O
O
N
O
O
+ RCHO + CO2
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27.10: Partial Hydrolysis of Peptides. Acidic hydrolysis ofpeptides cleave the amide bonds indiscriminately.
Proteases (peptidases): Enzymes that catalyzed the hydrolysisof the amide bonds of peptides and proteins.
Enzymatic cleavage of peptides and proteins at defined sites:• trypsin: cleaves at the C-terminal side of basic residues,
Arg, Lys but not His
• chymotrypsin: cleaves at the C-terminal side of aromatic residuesPhe, Tyr, Trp
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
NH3
NH
O
HN
O
H3N
R3
O
HN
R1O
H3NCO2
NH3
O +trypsin
H2O
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2 N
HO
HN
O
H3N
R3
O
HN
R1O
H3NCO2
O +chymotrypsin
H2O

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Trypsin and chymotrypsin are endopeptidases
Carboxypeptidase: Cleaves the amide bond of the C-terminal amino acid (exopeptidase)
27.11: End Group Analysis. The C-terminal AA is identified by treating with peptide with carboxypeptidase, then analyzingby liquid chormatography (AA Analysis).
N-labeling: The peptide is first treated with 1-fluoro-2,4-dinitrobenzene (Sanger’s reagent), which selectively reacts with the N-terminal amino group. The peptide is then hydrolyzed to theiramino acids and the N-terminal amino acid identified as itsN-(2,4-dinitrophenyl) derivative (DNP).
NO2
O2N
F
H3N
O
R1 HN CO2+
!
NH
O
R1 HN CO2nucleophilic
aromaticsubstitution
NO2
O2N
NH
O
R1
NH2
NO2
O2N
+plus other unlabeled
amino acids
enzymatic
digestion
-or-
H3O+, !
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27.12: Insulin. (please read) Insulin has two peptide chainsInsulin has two peptide chains (the A chain(the A chain has 21 amino acids and the B chain has 30 aminohas 21 amino acids and the B chain has 30 amino acids) held together by two disulfide linkagesacids) held together by two disulfide linkages
Pepsin: cleaves at the C-terminal side of Phe, Tyr, Leu; butnot at Val or Ala
Pepsin cleavageTrypsin cleavageH3O+ cleavage

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27.13: The Edman Degradation and Automated PeptideSequencing. Chemical method for the sequential cleavage andidentification of the amino acids of a peptide, one at a time starting from the N-terminus. Reagent: Ph-N=C=S, phenylisothiocyanate (PITC)
S
C
NPh
+H2N
O
R1 HN CO2
pH 9.0
NH
O
R1 HN CO2
NH
S
Ph
H+
H+
HN
S
OH
HN CO2
N
R1
PhH+
HN
S OH2N CO2
N
R1
Ph
H+
HN
N OS
R1
Ph
+
N-phenylthiohydantoin:separated by liquid chromatography (based of the R group) and detected by UV-vis
-1 peptide with a new N-terminal amino acid (repeat degradation cycle)
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Peptide sequencing by Edman degradation: • Cycle the pH to control the cleavage of the N-terminal amino acid by PITC. • Monitor the appearance of the new N-phenylthiohydantoin for each cycle. • Good for peptides up to ~ 25 amino acids long. • Longer peptides and proteins must be cut into smaller fragments before Edman sequencing.
Tandem mass spectrometry has largely replaced Edman degradation for peptide sequencing
27.14: The Strategy for Peptide Synthesis:Chemical synthesis of peptide:
1. Solution phase synthesis2. Solid-phase synthesis

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H2N CO2H
Ala
H2N CO2H
Val
+- H2O
H2N
HN
O
CO2H
Ala - Val(A - V)
H2N
HN
O
CO2H
Val - Ala(V - A)
- H2O
The need for protecting groups
Orthogonal protecting group strategy: the carboxylate protecting group must be stable to the reaction conditions for the removal of the α-amino protecting group and ( vice versa)
NH
Ala
H2N
Val
+
- H2O
NH
HN
O
Ala - Val(A - V)
Pn OH
O
OPc
O
PnOPc
Oselectivelyremove Pn
H2N
HN
O
Ala - Val(A - V)
OPc
O
peptidecoupling
peptidecoupling(-H2O)
NH
Pn OH
O
Ph
Phe (F)
NH
HN
O
OPc
OOHN
Ph
Pn
Phe - Ala - Val(F - A - V)
Repeat peptide synthesis
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27.15: Amino Group Protection. The α-amino group is protected as a carbamate.
27.16: Carboxyl Group Protection. Protected as a benzylester; removed by hydrogenolysis
RO Cl
O+ Base
NH
O
O
O
tert-butoxycarbonyl(t-BOC)
removed withmild acid
R
NH
O
O
O
C6H5
benzyloxycarbonyl(cBz)
removed with mild acid or by hydrogenolysis
R
NH
O
O
O
fluorenylmethylcarbonyl(FMOC)
removed with mild base(piperidine)
NH3
O
O NH
O
RO
O
OH
NH
H2N+- H2O
OH
O
O
O
peptidecoupling
O
OC6H5
C6H5NH
HN
O
O
O C6H5
O
OC6H5
mild acid
H2N
HN
O
O
O C6H5
OH
O
Ph
NH
O
OC6H5
- H2Opeptidecoupling
NH
HN
O
OOHN
Ph
O C6H5
O
OC6H5
H2, Pd/C
NH
HN
O
O
OO
H3N
Ph

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27.17: Peptide Bond Formation. Amide formation from the reaction of an amine with a carboxylic acid is slow. Amide bondformation (peptide coupling) can be accelerated if the carboxylicacid is activated. Reagent: dicyclohexylcarbodiimide (DCC)
C6H11 N C N C6H11
R O
O
C6H11 N C NH
C6H11
R O
O
+
R O
O
C
N
C6H11
C6H11
NH
R'-NH2
••
R O
O
C
HNC6H11
C6H11
NH
NHR' +
R NH
O
R'NH
NH
O
C6H11 C6H11+
(DCC)
Amide DCU
"activated acid"
R O
O
C
N
C6H11
C6H11
NH
NR'
++H
H
H
NH
Ala
H2N
Val
+NH
HN
OcBz OH
O
OBn
O
cBzOBn
O
selectivelyremove N-protecting
group
H2N
HN
O
OBn
O
NH
cBz OH
O
Ph
Phe (F)
NH
HN
O
OBn
OOHN
Ph
cBz
DCC
peptidecoupling
CF3CO2H
DCCH2, Pd/C
NH
HN
O
OH
OO
H2N
Ph
Phe - Ala - Val(F - A - V)
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• In order to practically synthesize peptides and proteins, time consuming purifications steps must be avoided until the very end of the synthesis.• Large excesses of reagents are used to drive reactions forward and accelerate the rate of reactions.• How are the excess reagents and by-products from the reaction, which will interfere with subsequent coupling steps, removed without a purification step?
27.18: Solid-Phase Peptide Synthesis: The Merrifield Method.Peptides and proteins up to ~ 100 residues long are synthesized on a solid, insoluble, polymer support. Purification is convenientlyaccomplished after each step by a simple wash and filtration.

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The solid support (Merrifield resin): polystyrene polymer
H2N
Val
O
O
peptidecoupling
NH
FMOC OH
ODCC
O
O
HN
FMOC NH
Opurify:
wash & filter
remove N-protecting
group
O
O
H2NNH
O
NH
FMOC OH
O
Ph
Phe (F)
DCC
O
O
NH
O
purify:wash & filter
NH
FMOCHN
O
Ph purify:wash & filter HF
remove N-protecting
group and cleavefrom solid-support
OH
O
NH
O
H2N
HN
O
Phpurify by liquidchromatograrphy
or electrophoresis
NH
remove N-protecting
group
NH
Solid-phase peptide synthesis
O
R
OHN_
O
O
R
NH
CF3CO2H
O
O
R
NH2
BOC
BOC
+
polymerization
styrene
divinylbenzene(crosslinker, ~1 %)
initiator
Ph
Ph Ph Ph Ph
PhPhPh Ph
Ph
Ph
Ph
Ph
Ph
H3COCH2ClZnCl2
CH2Cl
332
LYS GLU THR ALA ALA ALA LYS PHE GLU ARG GLN HIS MET ASP SER SER THR SER ALA ALA SER SER SER ASN TYR CYS ASN GLN MET MET LYS SER ARG ASN LEU THR LYS ASP ARG CYS LYS PRO VAL ASN THR PHE VAL HIS GLU SER LEU ALA ASP VAL GLN ALA VAL CYS SER GLN LYS ASN VAL ALA CYS LYS ASN GLY GLN THR ASN CYS TYR GLN SER TYR SER THR MET SER ILE THR ASP CYS ARG GLU THR GLY SER SER LYS TYR PRO ASN CYS ALA TYR LYS THR THR GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE ASP ALA SER VAL
His-12 AHis-119 A
His-12 BHis-119 B
pdb code: 1AFL
Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNASolid-phase synthesis of RNase A:
Synthetic RNase A: 78 % activity 0.4 mg was synthesized 2.9 % overall yield average yield ~ 97% per coupling step
R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry: “for his development of methodology for chemical synthesis on a solid matrix.”

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27.19: Secondary Structures of Peptides and Proteins.β-sheet: Two or more extended peptide chain, in which the amidebackbones are associated by hydrogen bonded
parallel
crossover
N
O
R
N
O
R
N
N
O
R
HN
O
R
N
O
R
O
R
N
R
O
O
H
H
H
H
H
H
N
O
R
N
O
R
N
N
O
R
HN
O
R
N
O
R
O
R
N
R
O
O
H
H
H
H
H
H
N → C
N → C
N → C
C ← N
anti-parallel loopor
turnN
O
R
N
O
R
N
N
O
R
N
O
R
N
O
R
O
R
N
R
O
O
H
H
H
H
H
H
N
O
N
O
R
N
N
O
R
N
O
R
N
O
R
O
R
N
R
O
O
H
H
H
H
H
H H
HN → C
N → C
C ← N
N → C
334
α-helix: 3.6 amino acids per coil, 5.4 Å
3.6 AA5.4 Å
C
N

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myoglobin pdb code: 1WLA
Anti-parallel β-sheets of lectin
pdb code: 2LAL
Bacteriorhodopsinpdb code: 1AP9
Parallel β-sheets carbonic anhydrase
pdb code: 1QRM
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27.20: Tertiary Structure of polypeptides and Proteins.Fibrous. Polypeptides strands that “bundle” to form elongated fibrous assemblies; insoluble;
Globular. Proteins that fold into a “spherical” conformation .
Hydrophobic effect. Proteins will fold so that hydrophobic amino acids are on the inside (shielded from water) and hydrophilic amino acids are on the outside (exposed to water).
Pro • Ile • Lys • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His • Ser • Lys

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Enzymes: proteins that catalyze biochemical reactions.• by bringing the reactive atoms together in the optimal geometry for the reaction.• lowering the activation energy (ΔG‡) by stabilizing the transition state and/or high energy intermediate. • many enzymes use the functional groups of the amino acid sidechain to carry out the reactions
Proteases (peptidases): catalyzes the hydrolysis of peptide bonds
NH
HN
NH
O
RO
HN
N
O R
RO
R
H
O protease
H2OH3N
CO2 NH
HN
H3N
O
RO
HN
N
O R
RO
R
H
O
H3NCO2O +
Four classes of proteases:Serine (trypsin): aspartate-histidine-serine Aspartyl (HIV protease, renin): two aspartatesCysteine (papain, caspase): histidine-cysteineMetallo (Zn2+) (carboxypeptidase, ACE): glutamate
338
Mechanism of carboxylpeptidase, metalloprotease (p. 1151)Mechanism of a serine protease (trypsin, chymotrypsin):
OSer195
H
N
NHis57
R1 NH
O
R2
NH HN
oxy-anionhole
H
Asp102 CO2-
R1 NH
O
R2
NH HN
OSer195
N
NHis57
H
Asp102 CO2-
H- R2-NH2
Ser192O R1
O
NH HN
acyl-enzymeintermediate
OH
H
N
NHis57
H
Asp102 CO2-
Ser192O R1
O
NH HN
OH
N
NHis57
H
Asp102 CO2-
H
OSer195
H
N
NHis57
H
Asp102 CO2-
RCO2H+

173
339
27.21: Coenzymes. Some reactions require additional organic molecules or metal ions. These are referred to as cofactors orcoenzymes.
N N
SOP
HO O
O POH
OH
O
NH2
N
+
Thiamin Diphosphate(vitamin B1)
OP
OHO
HO
N
O
OH
H
Pyridoxal Phophates(vitamin B6)
N
NN
N
O
HOO
OH
Fe
Heme
S
NHHN
O
CO2H
Biotin(vitamin B7)
NN
N N
H2N
O NH2
O
NH2
NH2O
O
O
Co
O
H
H2N
O
NH
O
C
PO
O
-OO
N
N
HO H
H
NH2
H
H
OH
N
Vitamin B12
(cyanocobalamin)
HN
N
N
N
HN
H2N
O
HN
O CO2-
CO2-
Folic Acid(vitamin B9)
N
N
NH
N O
O
O
OH
HO
OH
O-
P
O
P O
O
O-
O
HO
N
OH
O
N
N
N
NH2
Flavin Adenine Diphosphate (FAD)(Vitamin B2)
27.22: Protein Quaternary Structure. (please read)